Microorganisms are present ubiquitously in biological specimens and environmental media suitable for their growth. However, some prove harmful to higher organisms and means for detecting their presence is important to preserve the public health. Many means for detecting various types of microorganisms are available offering various advantages with respect to speed and specificity.
All microorganisms have certain requirements for growth and reproduction. In general, microorganisms require the presence of the following for growth: an energy source including light and carbon compounds; and a source of raw materials including carbon, nitrogen, sulfur, and phosphorous as well as trace amounts of other minerals. Further, microorganisms must be present in a suitable environment wherein an appropriate temperature, pH, salinity and oxygen concentration is maintained.
A common procedure used to detect the presence of microorganisms involves adding a specimen to a culture medium containing all the necessary elements to allow growth. The sample may be natural or pretreated, as by membrane filtration, before being added to the culture medium, and the medium may or may not contain chemicals such as antimetabolites or antibiotics which are selectively active against microorganisms other than the target microorganism. Usually, these culture media have been sterile to assure no interference from contaminating microbes, and usually a rather long incubation step of from 48 to 72 hours has been required to provide for appropriate detection of Enterococci. Additionally, once growth is detected in these procedures, the target microorganism must be confirmed using one or more of a number of tests specific for a variety of physical and biochemical characteristics. These procedures are therefore labor intensive and time consuming.
Many efforts have been made to simplify and expedite the detection processes. Among these efforts have been attempts to measure specific by-products of individual bacteria, electrical impedance assays, ATP assays, and carbon-14 labelled substrate assays. Most have not proven highly efficacious. Also, indicators of microbial growth have been used which change color only after the microbe grows. They normally react chemically with a metabolic by-product produced by the target microbes. Chemicals which change color in the presence of pH changes associated with growth including phenol red, bromocresol blue, and neutral red have also been used. For instance, Golber, U.S. Pat. No. 3,206,317 uses phenol red in the presence of an acidic medium produced by bacterial waste products. Berger et al., U.S. Pat. No. 3,496,066 describes the use of compounds which bacteria convert to dyestuffs. Bochner, U.S. Pat. No. 4,129,483 describes using a non-biodegradable substance which is reduced to produce a color change. In all of these situations, the indicator is an additional substance and not one which also serves as a source of a required nutrient.
Edberg, U.S. Pat. No. 4,925,789 describes the use of a nutrient indicator which not only serves as a nutrient source, but also changes color upon being metabolized. The patent, herein incorporated by reference, provides a medium containing a nutrient indicator which, when metabolized by a target bacteria, releases a moiety which imparts a color or other detectable change to the medium. The procedure takes advantage of enzyme specificity unique to particular species or groups of bacteria. It suggests using antibiotics to select for growth of the microorganisms targeted and provides specific examples of liquid-based assays. Other methods previously used such as Kilian et al., Acta. Path. Microbiol. Scand. Sect. B .sctn.7 271-276 (1979) and Damare et al., J. Food Science 50:1736 (1985) report use of agar-based media without antibiotics.
Enterococcus density is a predictor of public health risks associated with contaminated recreation waters. There are two accepted methods for the analysis of Enterococcus density in water samples, the multiple-tube for most probable number technique (MPN) and the membrane filter technique (MF) (Greenberg et al., Standard methods for the evaluation of water and wastewater Eaton, A.D. (ed.) 18th ed. American Public Health Association (1992); and Mooney, K. et al., Testing the waters: a national perspective on beach closings Natural Resources Defense Council. (1992)). The results based on the multiple-tube technique may not be available for 72 hours, and the results of the membrane filter technique may not be available for 48 hours. The "MPN procedure" involves a 24 to 48 hour presumptive test in a series of azide dextrose broth followed by a 48 hour confirmation test using selective Enterococcus agar and 6.5% NaCl brain-heart infusion broth. The membrane filter technique involves the membrane filtration of water samples followed by incubation of a pre-filtered sterile membrane on Enterococcus selective media. The media of choice are either mE agar followed by an EIA substrate test, or menterococcus agar. Such methods may be tedious, labor intensive and time consuming. This may lead to delays in public notification and therefore increase public health risks.